Spontaneous cellular electrical rhythms (or pacing) govern numerous biological processes from the autonomous beating of the heart, pain transmission, to respiratory rhythms and insulin secretion. For instance, spontaneous neuronal electrical discharges in damaged dorsal root ganglions underlie neuropathic pain but limited useful therapy is available. Recently, we have also discovered that key ionic components which underlie the electrical rhythms in pancreatic β cells also modulate the secretion of insulin. In the heart, abnormal pacing leads to various forms of arrhythmias and electrical disorders that necessitate traditional pharmacologic interventions and implantation of costly electronic devices that are associated with various side effects, inherent risks, and expenses.
Autonomous rhythmic heart beats are modulated by sympathetic and parasympathetic means according to everyday needs; such normal rhythms originate in the sino-atrial (SA) node of the heart, a specialized cardiac tissue consisting of a few thousands pacemaker cells that spontaneously generate rhythmic action potentials (AP) (i.e. pacing). One of the key players known to prominently modulate the pacing activity of SA nodal cells is the cardiac membrane current If (“f” for funny), a depolarizing, mixed Na+/K+ inward current1. Despite the fact that If has been recognized for over 20 years, the encoding genes, collectively known as the hyperpolarization-activated cyclic-nucleotide-modulated (HCN) channel gene family, have been cloned relatively recently.2-4 To date, four isoforms, namely HCN1-4, each with a distinct pattern of tissue distribution and biophysical profiles, have been identified3-8. Of the two predominant isoforms in the SA node, time-dependent HCN1 currents open ˜40 times faster than those of HCN4 channels9-13; a single base-pair deletion mutation detected in the human HCN4 gene of a patient has been linked to idiopathic sinus node dysfunction14.
Since HCN1-4 readily co-assemble to form heterotetrameric complexes with context-dependent properties that can not be predicted from the individual isoforms15-18, native If can have complex molecular identity depending upon the particular isoforms expressed. Furthermore, HCN channels activate more positively in native cardiomyocytes than in mammalian expression systems, suggesting that the gating properties of If are highly context-dependent (Qu et al 2002 Pflugers) (e.g. the presence of endogenous subunits). Thus, native If is difficult to reproduce by simple expression of a single HCN isoform. Indeed, previous attempts to overexpress wild-type HCN2 in adult ventricular cardiomyocytes failed to induce automaticity19, presumably due to its negative activation relative to the voltage range of cardiac pacing. It would be highly desirable to develop a flexible and effective approach that enables us to delicately customize the activity of HCN channels so as to achieve a range of therapeutic outcomes. For instance, to engineer a HCN channel construct, which opens more readily than wild-type HCN channels (and thereby compensates the context-dependent negative activation shift in heart cells) to better mimic native nodal If so as to effectively induce or modulate cardiac automaticity. The same principle can be extrapolated for application in other cell types whose functions depend on electrical rhythms.
As previously mentioned, HCN-encoded If (or Ih) plays an important role in the spontaneous rhythmic activity in cardiac, neuronal as well as insulin-secreting cells (32-38). Although classical depolarization-activated voltage-gated K+ (Kv) and HCN channels are structurally homologous to each other, the latter are uniquely distinctive from the Kv counterparts by their signature ‘backward’ gating (i.e. activation upon hyperpolarization rather than depolarization). The basis of HCN gating is largely unknown.
Recent evidence suggests that the voltage-sensing mechanisms of HCN and Kv channels are conserved despite their opposite gating behaviors (i.e. the HCN S4 also moves outward and inward during depolarization and hyperpolarization, respectively) (39,40). This finding raises the possibility that the S3-S4 linker (defined as residues 229EKGMDSEVY237 of HCN1; FIG. 1), which is directly tethered to the S4 voltage-sensor, also influences the activation phenotypes of HCN channels as does that of Kv channels (41-43). Indeed, we have recently reported that the S3-S4 linker contains several functionally-important residues (44, 45). For instance, single alanine substitutions of G213, M232 and E235 produced depolarizing activation shifts. The pattern of site-dependent perturbations of HCN activation, along with computational modeling, further suggests that part of the linker conforms a helical secondary structure with the determinants G231, M232 and E235 clustered on one side. It would be desirable to understand the structural and functional roles of the HCN S3-S4 linker. Such understanding would be helpful in developing engineered HCN channels which would open more (or less) readily for increasing (or decreasing) automaticity in cardiac, neuronal, pancreatic cells, etc.
Throughout this application, various publications are referenced to by numbers. Full citations for these publications may be found at the end of the specification immediately following the Abstract. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to those skilled therein as of the date of the invention described and claimed herein.